Meganuclease variants cleaving a dna target sequence from the hprt gene and uses thereof

Abstract

A method for inducing a site-specific modification in the HPRT gene, for a non-therapeutic purpose, by contacting a DNA target sequence selected from the group consisting of the sequences SEQ ID NO: 1 to 14 thereby cleaving the DNA target with an I-CreI variant or single-chain derivative having at least one substitution in one of the two functional subdomains of the LAGLIDADG (SEQ ID NO: 153) core domain situated from positions 26 to 40 and 44 to 77 of I-CreI.

1 . A method for inducing a site-specific modification in the HPRT gene, comprising contacting a DNA target sequence selected from the group consisting of the sequences SEQ ID NO: 1 to 14 thereby cleaving the DNA target with an I-CreI variant or single-chain derivative having at least one substitution in one of the two functional subdomains of the LAGLIDADG (SEQ ID NO: 153) core domain situated from positions 26 to 40 and 44 to 77 of I-CreI. 
     
     
         2 . The method according to  claim 1 , wherein said HPRT gene is a non-human mammal HPRT gene. 
     
     
         3 . The method according to  claim 2 , wherein said HPRT gene is the  Criteculus  sp. HPRT gene. 
     
     
         4 . The method according to  claim 2 , wherein said HPRT gene is the  Mus musculus  HPRT gene. 
     
     
         5 . The method according to  claim 4 , wherein said DNA target sequence is of the sequence SEQ ID NO: 6, 7, 8, 9, 10, 11, 12 or 14. 
     
     
         6 . The method according to  claim 1 , wherein said HPRT gene is the  Homo sapiens  HPRT gene. 
     
     
         7 . The method according to  claim 6 , wherein said DNA target sequence is of the sequence SEQ ID NO: 6, 8, 9, 12 or 14. 
     
     
         8 . The method according to  claim 1 , wherein said I-CreI variant or single-chain derivative is combined with a targeting DNA construct comprising a sequence to be introduced flanked by sequences sharing homologies with the regions of the HPRT gene surrounding the genomic DNA cleavage site of said I-Cre I variant or single chain derivative. 
     
     
         9 . The method according to  claim 8 , wherein said sequence to be introduced comprises a gene of interest. 
     
     
         10 . The method according to  claim 8 , wherein said sequence to be introduced comprises an inactivation cassette for the HPRT gene. 
     
     
         11 . The method according to  claim 8 , wherein said targeting DNA construct is inserted in a vector. 
     
     
         12 . The method according to  claim 1 , wherein said I-CreI variant or single-chain derivative is encoded by a polynucleotide fragment. 
     
     
         13 . The method according to  claim 12 , wherein said fragment is inserted in an expression vector. 
     
     
         14 . The method according to  claim 13 , wherein said vector comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric I-Cre I variant. 
     
     
         15 . The method according to  claim 13 , wherein said vector includes a targeting DNA construct comprising a sequence to be introduced flanked by sequences sharing homologies with the regions of the HPRT gene surrounding the genomic DNA cleavage site of said I-Cre I variant or single chain derivative. 
     
     
         16 . The method according to  claim 1 , wherein said site-specific modification of the HPRT gene consists in the insertion of a gene of interest by cleavage of the HPRT gene by said I-CreI variant and homologous recombination with a targeting DNA construct comprising a gene of interest. 
     
     
         17 . The method according to  claim 1 , wherein said site-specific modification of the HPRT gene consists in the insertion of an inactivation cassette by cleavage of the HPRT gene by said I-CreI variant and homologous recombination with a targeting DNA construct comprising an inactivation cassette for the HPRT gene. 
     
     
         18 . The method according to  claim 1 , wherein said site-specific modification of the HPRT gene consists in the inactivation of the HPRT gene by cleavage of the HPRT gene by said I-CreI variant and repair of the double-strands break by non-homologous end joining. 
     
     
         19 . The method according to  claim 1 , wherein said substitution(s) in the subdomain situated from positions 44 to 77 of I-CreI are in positions 44, 68, 70, 75 and/or 77. 
     
     
         20 . The method according to  claim 1 , wherein said substitution(s) in the subdomain situated from positions 26 to 40 of I-CreI are in positions 26, 28, 30, 32, 33, 38 and/or 40. 
     
     
         21 . The method according to  claim 1 , wherein said I-CreI variant comprises one or more additional substitution(s) in I-CreI. 
     
     
         22 . The method according to  claim 21 , wherein said substitutions are at positions: 2, 9, 19, 42, 43, 54, 66, 69, 72, 81, 82, 86, 90, 92, 96, 100, 103, 104, 105, 107, 108, 109, 110, 113, 120, 125, 129, 130, 131, 132, 135, 136, 137, 140, 143, 151, 154, 155, 157, 158, 159, 161 or 162 of I-CreI. 
     
     
         23 . The method according to  claim 22 , wherein said substitution is the G19S or G19A mutation. 
     
     
         24 . The method according to  claim 1 , wherein said I-CreI variant is an heterodimer, resulting from the association of a first and a second monomer having different mutations in positions 26 to 40 and/or 44 to 77 of I-CreI. 
     
     
         25 . The method according to  claim 24 , wherein at least one monomer has at least two substitutions, one in each of the two functional subdomains situated from positions 26 to 40 and 44 to 77 of I-CreI. 
     
     
         26 . The method according to  claim 25 , wherein said heterodimer consist of a first and a second monomer selected from the following pairs of sequences: SEQ ID NO: 83 and 97, SEQ ID NO: 84 and 98, SEQ ID NO: 85 and 99, SEQ ID NO: 32 and 52, SEQ ID NO: 32 and 53, SEQ ID NO: 32 and 54, SEQ ID NO: 32 and 55, SEQ ID NO: 32 and 56, SEQ ID NO: 32 and 57, SEQ ID NO: 32 and 58, SEQ ID NO: 32 and 60, SEQ ID NO: 32 and 65, SEQ ID NO: 32 and 66, SEQ ID NO: 32 and 67, SEQ ID NO: 32 and 68, SEQ ID NO: 32 and 69, SEQ ID NO: 32 and 70, SEQ ID NO: 32 and 71, SEQ ID NO: 32 and 72, SEQ ID NO: 32 and 73, SEQ ID NO: 32 and 74, SEQ ID NO: 75 and 56, SEQ ID NO: 76 and 56, SEQ ID NO: 77 and 56, SEQ ID NO: 78 and 56, SEQ ID NO: 79 and 56, SEQ ID NO: 80 and 56, SEQ ID NO: 81 and 56, SEQ ID NO: 82 and 56, SEQ ID NO: 86 and 96, SEQ ID NO: 87 and 100, SEQ ID NO: 88 and 101, SEQ ID NO: 89 and 102, SEQ ID NO: 90 and 103, SEQ ID NO: 91 and 104, SEQ ID NO: 92 and 105, SEQ ID NO: 93 and 106, SEQ ID NO: 94 and 107, SEQ ID NO: 95 and 108, and SEQ ID NO: 147 and 148. 
     
     
         27 . The method according to  claim 24 , wherein one monomer of the heterodimer comprises the G19S mutation.